Detectors used in analytical methods and screening protocols are often nonspecific, require complex chromogenic interactions, or are not well adapted to microscale systems. Analytical systems typically employ spectrophotometers, conductivity meters, pH meters, and the like, to detect elution of analytes from chromatographic separations. More specific detection of analytes often requires complex layered systems of affinity molecules, enzymes and substrates. Available detection systems often employ optical detection schemes for chemically labeled components which can suffer from excessive background signal, nonspecific signals, difficult data interpretation, analyte specific chemistry requirements, and the like.
Spectrophotometry can be useful to detect biological molecules in analytical systems. Proteins and nucleic acids have peak absorbances near the ultraviolet region for detection using photomultiplier tubes or photodiode array sensors. Such sensors can be blanked (set to zero) with analytical buffer in the flow cell. As the biological molecule elutes from a chromatographic separation (such as ion exchange, RP-HPLC, SEC, and the like) and flows into the flow cell, an absorbance peak can be detected by a spectrophotometer. Such spectrophotometric detection is generally nonspecific, can be expensive, and often yields relatively high signal to noise ratios, particularly when detecting very small concentrations of analyte.
Spectrophotometry can also be used to identify specific molecules in arrays or chromatographic eluates. In one common format, a biologic molecule is bound to a molecule with specific affinity (such as an antibody, lectin, receptor, or the like) which is also bound to a detectable marker (such as a radionuclide, an enzyme, or a fluorescent tag). The complex “sandwich” of bound molecules can be specifically detected by spectroscopic instruments. These affinity based systems can provide specific detection of molecules, but in addition to the above-mentioned difficulties, can also require the use of hazardous materials and complex plumbing for assay automation.
Conductivity and pH meters are commonly used to monitor chromatographic processes and analytical elutions. Such detectors can be configured to work in processing equipment, analytical instrumentation, or microfluidic devices to monitor the passage of charged molecules or buffer solutions. These detectors are typically not useful in detection of biological molecules for lack of specificity and sensitivity. In addition, the high impedance required for many of these detectors to sense changes in microenvironments can provide a noisy signal with poor sensitivity.
Biosensors have been described in which a mass of bound analyte molecules can change the resonant vibration frequency of a micro beam. In U.S. Pat. No. 6,303,288, Integrated Microchip Genetic Testing System, to Furcht, for example, molecular binding sites are located on the surface of a microbeam with a known vibration frequency. When molecules bind to the sites, the mass of the microbeam increases, reducing the vibrational frequency of the microbeam. Such vibrations can be induced and detected, e.g., as voltage potentials associated with a piezo-electric microbeam component. Another aspect of Furcht is detection of mechanical stress on an underlying piezoelectric element when analytes load onto the binding sites. Beam style piezoelectric detection devices require a complex multilayered etching and coating technology, produce microscale (but not nanoscale) sensor elements, and can have a disadvantageous ratio of binding surface to sensor mass.
Chemical sensors have also been described that employ binding agents on the surface of a semiconductor channel that connects a source and drain electrode. The binding of large amounts of charged analyte to the surface of the channel cases an electric field induced gating of the channel. While such chemically sensitive field effect transistors (or ChemFETs) have shown some functionality, the requirement of very large concentrations of charged analyte to achieve a detectable gating of the transistor has resulted in their not being particularly useful for most applications. Lieber et al., recently reported a ChemFET (see U.S. Patent Application number 2002/0117659, “Nanosensors”, and Published International Patent Application number WO 02/48701, each of which is incorporated herein by reference in its entirety for all purposes) operating on the same basic principles, but which employs a semiconductor nanowire as the channel component. These nanoChemFETs reportedly have dramatically increased sensitivity as compared to the previously reported ChemFETs theoretically as a result of their substantially increased surface area to volume or cross-section ratio, e.g., providing a much larger binding region with a much smaller channel cross-section that is to be gated by the binding event. Despite these reported advances, there are a number of areas that would be ripe for improvement of ChemFET and nanoChemFET based devices and systems.
In particular, there remains a need for microsensors and/or nanosensors, as well as methods of utilizing such sensors, that combine the sensitivity of nanoChemFET based sensors with the robustness and data reliability of more conventional systems. The present invention provides these and other features which will be apparent upon complete review of the following.